Cell & Molecular Imaging Core D ?John Lemasters, MD, PhD, Core Leader Abstract Cell & Molecular Imaging Core D provides major support in confocal, multiphoton and brightfield microscopy to COBRE investigators. The Imaging Core houses the following major microscope systems: 1) a Zeiss LSM 880 NLO confocal/multiphoton microscope equipped with a Coherent Chameleon multiphoton laser with Quasar photomultiplier array for spectral analysis; 2) an Olympus FV1200 multiphoton microscope with Spectra- Physics MaiTai multiphoton laser and silicone oil optics for intravital imaging, 3) an Olympus FV10i LIV live cell confocal microscope with water immersion optics, 4) a Zeiss LSM 510 META laser scanning confocal microscope; and 5) a BD CARV II disk-scanning confocal microscope for video-rate imaging as well as conventional widefield microscopy. All microscopes are equipped with environmental chambers for temperature and gas phase control to allow high resolution non-destructive 3-dimensional imaging of living cells, tissues and organisms. Major applications of this instrumentation include 1) live cell imaging of parameter-sensitive fluorophores to monitor ions, electrical potentials, oxygen and nitrogen radical generation, pyridine nucleotide reduction, mitochondrial and plasmalemmal membrane permeability, cell viability (apoptosis and necrosis), fluorescent protein labeling and other parameters; 2) intravital microscopy to monitor microcirculation, leukocyte margination, mitochondrial polarization and permeability, radical generation, gene expression and other parameters in living animals; 3) fluorescence and brightfield imaging of immuno-stained tissue samples; 4) fluorescence resonance energy transfer (FRET) to characterize and quantify interactions between specific molecules; 5) Duolink proximity ligation assay to study interactions between endogenously expressed proteins. Ancillary equipment includes a digital darkroom facility for digitizing, analyzing and labeling images; software and computer workstations for 3- and 4-D image analysis and volume rendering; tissue culture hoods and incubators for specimen preparation; and fluorescence and absorbance plate readers for parallel measurements in cultured cells grown on multi-well plates. Imaging core services will promote the success of the individual COBRE projects and will also provide training and assistance to junior investigators studying oxidative stress and stress signaling related to the overall theme of this COBRE. In Phase 1 of the COBRE, CMI Core D contributed to 29 of 44 publications and 26 of 35 grant applications by COBRE investigators.